The Tight Junction (TJ)
In multicellular organisms fluids with different molecular compositions (urine, milk, gastric juice, blood etc.) are contained in compartments delineated by epithelia (e.g. renal tubules) and endothelia (blood vessels). These cellular sheets constitute the frontier between the organism internal milieu and the compartments' contents. Therefore in order for components of the blood to enter a given tissue, they must first traverse from the lumen of the blood vessel through the endothelial cells of that vessel. In case of substances that enter the body via the gut, they must first pass the barrier formed by the epithelial cells that line the cavity, and to enter the blood via the skin, both epithelial and endothelial sheets must be crossed.
Although cell-cell adhesion is crucial to develop tissues and for maintaining discrete compartments within the organism, there are conditions in which a controlled regulation of cell adhesion is desirable. Such situation is encountered when the barrier formed by the epithelia or endothelia creates difficulties for the delivery of drugs to specific tissues and tumors within the body.
The passage of substances through endothelia and epithelia proceeds through two parallel routes: a transcellular and a paracellular pathway. In the former ions and molecules employ for their transit channels, carriers and pumps located in the plasma membrane of epithelia and endothelia. Attempts to facilitate the passage of drugs to specific tissues within the body have generally relied on such specific channels or carriers that in vivo transport molecules. However such methods have been largely inefficient due to low endogenous transport rates or to their poor functioning with applied drugs.
To overcome these impediments transport through the paracellular pathway has been assayed. This route consists of the intercellular space existent between adjacent cells and is regulated by the tight junction (TJ).
The TJ is a structure that surrounds the cellular borders at the limit between the apical and lateral membranes. It displays two fundamental roles: 1) as a gate that regulates the passage of ions, water and molecules through the paracellular route; and 2) as a fence that blocks the lateral diffusion within the plane of the membrane of lipids and proteins. This fence is crucial since it maintains the polarized distribution of lipids and proteins between the apical and basolateral to plasma membrane (Cereijido et al., 1998).
On ultrathin section electron micrographs, TJ are viewed as a series of fusion points “kisses” between the outer leaflets of the membranes of adjacent cells. At these kissing points, the intercellular space is completely obliterated. On freeze-fracture replica electron micrographs TJ appear at the plasma membrane as a network of continuous and anastomosing filaments on the protoplasmic face (P), with complementary grooves on the exoplasmic face (E) (Gonzalez-Mariscal et al., 2001).
Two models have been proposed to explain the chemical nature of TJ. In the protein model, TJ strands are formed by integral membrane proteins that associate with a partner in the apposing membrane of the adjacent cell. In the lipid model instead, TJ filaments are supposed to be formed of inverted cylindrical micelles (Kachar et al., 1982). Although the lipid content of the bilayer appears to be important for the formation of TJ, the discovery in recent years of TJ specific integral proteins gives strong support to the protein model of TJ.
TJ are constituted by a complex array of cortical and integral proteins. Of the former, 16 different molecules have so far been identified. Some function as scaffolds, that link the integral proteins of the TJ to the actin cytoskeleton (ZO-1, ZO-2, ZO-3 and cingulin) (Citi et al., 1988; Gonzalez-Mariscal et al., 2000), or as crosslinkers of transmembrane junctional proteins (MUPP1, MUPP2 and MUPP3) (Hamazaki et al., 2002). Others are involved in vesicular trafficking to the TJ (Rab13, Rab3b) (Zahraoui et al., 1994), in cell signaling through their association to kinases (Par3 and Par 6) (Izumi et al., 1998) and Ras (e.g. AF6) (Yamamoto et al., 1997), and in gene expression by their specific binding to transcription factors (ZO-1 and ZO-2) (Balda et al., 2000). The role of several other cortical proteins found at the TJ still remains unclear [e.g. Jeap (Nishimura et al., 2002), Pilt (Kawabe et al., 2001), Barmotin (Zhong et al., 1993) and symplekin (Keon et al., 1996)].
At the TJ three integral proteins are found: occludin, claudins and JAM. Occludin was the first one identified (Furuse et al., 1993). It is considered a component of TJ strands, since immuno replica electron microscopy with specific antibodies revealed its labeling within the TJ filaments (Saitou et al., 1997). Furthermore, when introduced into L fibroblasts, that lack TJ, structures that resemble TJ strands were formed.
Occludin comprises four transmembrane regions, two extracellular loops of similar size, and three cytoplasmic domains: one intracellular short turn, a small amino terminal domain and a long carboxyl terminal region.
Several lines of evidence assign occludin an important role at TJ. Thus, the over-expression of mutant forms of this protein in epithelial cells leads to changes in the barrier and fence function of TJ (Balda et al., 1996b; McCarthy et al., 1996) (Bamforth et al., 1999) and in the transepithelial migration of neutrophils (Huber et al., 2000). (Lacaz-Vieira et al., 1999). (Medina et al., 2000) (Vietor et al., 2001) Additionally, a correlation has been observed in several tissues between the expression of occludin and the degree of sealing of epithelia evaluated by transepithelial electrical resistance (TER) and permeability to extracellular tracers. Despite this evidence the physiological function of occludin is not completely understood. In this regard it should be highlighted that embryonic cells and mice carrying a null mutation in the occludin gene are still able to form well developed TJ (Saitou et al., 1998), although the animals display postnatal growth retardation and histological abnormalities in several tissues (Saitou et al., 2000).
More recently other integral proteins named claudin 1 and claudin 2 were identified as TJ constituents. By data base searching and cDNA and genomic cloning the claudin family has expanded to 20 members (Tsukita et al., 2001). All claudins encode 20 to 27 kDa proteins with four transmembrane domains; two extracellular loops where the first one is significantly longer than the second one, and a short carboxyl intracellular tail.
When claudins were transfected into fibroblasts, they conferred cell-cell aggregation activity, concentrated at the cells contact points and formed networks of filaments that looked like TJ strands. Furthermore, in immunoreplica electron microscopy antibodies against different claudins selectively labeled the TJ filaments of epithelia. All this evidence has let to consider claudins as the backbone of TJ strands.
Different claudin species are capable of generating different freeze fracture patterns. Thus, claudins 1 or 3 form TJ with continuous smooth fibrils on the protoplasmic surface (P face) of the replicas (Furuse et al., 1999), whereas claudins 2 or 5 generate junctions with discontinuous chains of particles associated to the exoplasmic face (E face) (Morita et al., 1999b). Claudin 11 instead constitutes parallel TJ strands on the P face that scarcely branch (Morita et al., 1999a).
Heterogeneous claudins can interact within a single TJ strand and their particular combination gives rise to different freeze fracture patterns. Thus strands formed with claudins 1 and 3 are continuous and associated to the P face, while strands formed with claudins 1 and 2 or 3 and 2 have evenly scattered particles in the E face grooves. At the paracellular space the extracellular loops of different species of claudins belonging to neighboring cells can also interact, except in some combinations (Furuse et al., 1999).
The expression of different claudins in epithelia and endothelia might give rise to the ample variety in permeability and paracellular ionic selectivity displayed in distinct tissues. The nephron that displays a wide range of TER along the different tubular segments (6 Ωcm2 in proximal Vs 870-2000 Ωcm2 in collecting duct) expresses almost all claudins, yet each one is restricted to a particular segment (Enck et al., 2001; Kiuchi-Saishin et al., 2002) (Reyes et al., 2002): claudins 5 and 15 at endothelia, claudins 2, 10 and 11 at the proximal segment, claudins 1, 3 and 8 at de distal tubule and claudins 1, 3, 4 and 8 at the collecting segment.
The onset of expression for different claudins is developmentally regulated. Thus, claudin 5 is transiently expressed during the development of the retinal pigment epithelium (Kojima et al., 2002), claudin 11 is expressed in Sertoli cells, immediately after the peak of expression of the sex determining region in the Y gene (Hellani et al., 2000), and claudin 6 is found in embryonic stem cells committed to the epithelial fate (Turksen et al., 2001).
Claudin 16 is mutated in human patients with hypomagnesemia hypercalciuria syndrome (HHS) (Simon et al., 1999). These patients manifest a selective defect in paracellular Mg2+ and Ca2+ reabsorption in the thin ascending limb of Henle's, with intact NaCl resorption ability at this site (Blanchard et al., 2001). Therefore claudin 16 appears to function as a paracellular channel selective for Mg2+ and Ca2+ (Goodenough et al., 1999). Other claudins are also proved ionic selective. Thus when claudin 4 is transfected into epithelial cells, the paracellular conductance decreases through a selective decrease in Na+ permeability without a significant effect on Cl− permeability (Van Itallie et al., 2001).
More than two decades ago Claude observed that the TER increases with the number of TJ strands, not in a linear fashion as would be expected from the addition of resistors in series, but exponentially (Gonzalez-Mariscal et al., 2001). To explain this relationship a proposal arose suggesting the existence of ion channels or pores within the TJ strands (Claude 1978; Gonzalez-Mariscal et al., 2001). Now that claudins have begun to be characterized, it appears that the ionic selectivity at the TJ could be determined by the specific claudin isoforms that constitute the pores or channels of TJ strands. On analyzing the extracellular loops of claudins an enormous variability in distribution and number of charged residues is found. For example the isoelectric points of the first loop range from 4.17 in claudin 16 to 10.49 in claudin 14, and in the second extracellular loop from 4.05 in claudins 2, 7, 10 and 14 to 10.5 in claudin 13. Based on the pKIs of the extracellular loops sequences, claudin 16 is a cation pore, a proposal that agrees with the observed effect of its mutation in human patients, whereas claudins 4, 11 and 17 are anion pores (Mitic et al., 2001).
Variations in the tightness of the TJ appear to be determined by the combination and mixing ratios of different claudin species. Thus when MDCK cells expressing claudin 1 and 4 were incubated with a claudin 4 binding peptide (Clostridium perfringens enterotoxin, CPE), claudin 4 was selectively removed from TJ, generating a significant decrease in TER (Sonoda et al., 1999). Furthermore, when claudin-2 was introduced into high resistance MDCK cells (MDCK I), TJ became leaky and morphologically similar to those found in low resistance cells (MDCK II), which normally contain claudin 2 (Furuse et al., 2001).
The role of claudins in carcinogenesis is controversial. Claudin 4 is over-expressed in pancreatic cancer and gastrointestinal tumors, and the treatment with TGFβ or CPE, the enterotoxin that specifically targets claudin 4, leads to a significant reduction of tumor growth (Michl et al., 2001). In contrast, other claudins remain low or undetectable in a number of tumors and cancer cell lines. For example Claudin 1 expression is lost in most human breast cancers without presenting alterations in its promoter or coding sequences (Hoevel et al., 2002; Kramer et al., 2000), and claudin 7 is downregulated in head and neck squamous cell carcinomas (Al Moustafa et al., 2002).
The crucial role of certain claudins in the gate function of epithelia is highlighted by the observation that in claudin 1 deficient mice the epidermis looses it barrier function, leading to dehydration of the animals, wrinkled skin and death within 1 day of birth (Furuse et al., 2002). In these mice occludin was still expressed at all layers of the stratified epithelia, and claudin 4 remained concentrated at the second and third layers of the stratum granulosum. Therefore in the epidermis claudin-1 constitutes an indispensable element for the barrier function of TJ.
The last integral proteins of the TJ are JAM and the three JAM like proteins (Palmeri et al., 2000). They belong to the immunoglobulin superfamily, have a single transmembrane segment and their extracellular portion consists of two folded immunoglobulin like domains. JAM appears not to be a constituent of TJ strands since its transfection into fibroblasts does not generate the appearance of filaments. JAM plays a role in cross-linking occludin and claudins, as well as in the transepithelial and transendothelial migration of monocytes (Martin-Padura et al., 1998).
Physiological and Pathological Regulation of Tight Junctions.
Epithelia and endothelia encounter diverse physiological and pathological conditions that provoke changes in the degree of sealing of TJ. These variations in TJ permeability are regulated by a broad spectrum of factors such as calcium (Gonzalez-Mariscal et al., 1990; Martinez-Palomo et al., 1980), hormones, cytokines and growth factors, activation of G proteins and phospholipases, generation of CAMP and diacylglicerol (Balda et al., 1991), and by the phosphorylation of TJ proteins by different kinases (Avila-Flores et al., 2001; Balda et al., 1996a; Sakakibara et al., 1997).
In recent years the action of enteric pathogens (e.g. Escherichia coli, Salmonella typhimurium) and bacterial toxins upon TJ has been recognized (Hecht2002). Thus, treatment with Clostridium perfringens enterotoxin (CPE) destroys TJ and TER by specifically removing claudins 3 and 4 from the strands (Sonoda et al., 1999), while the hemaglutinin and ZOT toxin of Vibrio cholera increase epithelial permeability due to their respective action upon occludin (Wu et al., 2000) and PKC (Fasano et al., 1995). From an evolutionary point of view, bacteria that counted with toxins that mimicked endogenous modulators of TJ were in advantage since by traversing epithelial barriers they gained access to new environments. This appears to be the case for Vibrio cholera, as an endogenous protein that modulates TJ has been recently identified employing antibodies generated against ZOT toxin (Fasano1999; Wang et al., 2000).
Rotaviruses
Rotaviruses are the leading cause of morbidity and mortality caused by gastroenteritis in children less than 2 years old. These viruses of the Reoviridae family have a genome composed of 11 double stranded segments of RNA, surrounded by three concentric layers of protein. The outermost layer is smooth and formed by a 37 kDa glycoprotein named VP7. From it around 60 spikes formed of an 88 kDa protein named VP4, project outwards (Estes1996).
VP4 is essential for early virus-cell interactions, since it participates in receptor binding and cell penetration. In fact the infectivity of rotaviruses is dependent upon the specific cleavage by trypsin of VP4 into peptides VP5 and VP8 (Almeida et al., 1978; Espejo et al., 1981).
In vivo rotavirus infection is restricted to ileum microvellosities (Kapikian et al., 1996). In vitro infectiveness is less restrictive as a broad variety of renal and intestinal epithelial cell lines are susceptible to rotavirus infection (Estes et al., 1989).
Some rotaviruses bind to a cell-surface receptor containing sialic acid (SA), while others (e.g. human rotavirus) do not require SA for infection (Fukudome et al., 1989). Therefore binding to SA appears not to be an essential step for rotavirus infection. Furthermore, association to a secondary SA independent receptor can overcome the initial interaction of certain rotavirus with SA. SA dependent rhesus rotavirus (RRV) initially bind to the cell through VP8 (Fiore et al., 1991; Isa et al., 1997), while variants of RRV which no longer depend on the presence of SA (e.g. nar3), interact with the cell through VP5 (Zarate et al., 2000b). The comparative characterization of many strains of animal and human origin, including RRV, its SA independent variant nar3, and the human rotavirus strain Wa, has shown that rotavirus contain integrin ligand sequences (Coulson et al., 1997) (Guerrero et al., 2000) and that α2β1 integrin is used as a primary cell receptor by nar3, and by RRV in a secondary interaction, subsequent to its initial contact with the SA containing cell receptor (Zarate et al., 2000a). Integrin αVβ3 is used by all three rotavirus strains as a co-receptor, subsequent to their initial attachment to the cell (Guerrero et al., 2000). Integrins αXβ2 and α4β1 have also been suggested to participate in rotavirus cell entry (Coulson et al., 1997; Hewish et al., 2000).
Aside of their function as cellular receptors for viruses, integrins constitute a family of αβ heterodimers that mediate the interaction between the cell and the extracellular matrix. This interaction plays a crucial role in the regulation of cell no proliferation, migration and differentiation. In epithelial and endothelial cells integrins have a polarized distribution and localize at the basolateral plasma membrane. Therefore rotaviruses contained in the lumen of the intestine or at the apical surface of confluent epithelial cell lines could only have access to their integrin receptors at the basolateral surface if the TJ that seal the paracellular route are opened.
Since as stated at the beginning of this description, there is a need in the art for compounds that modulate junctional tightness and improve drug delivery across permeability barriers, we proceeded to explore the capacity of rotavirus proteins to modulate TJ sealing. The present invention fulfills this need and provides other related advantages. Recently, cell transformation has been correlated with over expression of certain claudins (Michl et al., 2001). Therefore the rotavirus proteins and derived peptides that target junctional proteins could also be employed for reducing tumor cell growth.
In the present invention we have worked with proteins and peptides derived from the VP4 molecule of Rhesus monkey rotavirus (RRV). Rotavirus infect a wide variety of vertebrates, such as chickens, horses, pigs, monkeys and humans, and several strains of viruses have been isolated from different individuals of the same specie. However, since the amino acid sequence of VP4 maintains a high degree of identity among most of the different rotavirus strains, it is expected that the different strains independent of their origin will exert a similar effect upon TJ. In consequence in the present invention we will further refer to VP4 and its derived peptides, without placing special emphasis on their origin.